This invention relates to a new use of macrolide compounds for inducing chondrogenic differentiation.
The inventors of this invention have surprisingly found that the macrolide compounds mentioned here-in-below has an inducing activity of chondrogenic differentiation.
Accordingly, this invention provides a new use of the macrolide compounds for inducing chondrogenic differentiation.
Further, this invention provides an agent for inducing chondrogenic differentiation, which comprises the macrolide compounds.
Still further, this invention provides a method for inducing chondrogenic differentiation, which comprises administering said macrolide compounds to mammals.
Still further, this invention provides a method for preventing or treating damages of cartilage, which comprises administering said macrolide compounds to mammals.
The term xe2x80x9cmacrolide compoundxe2x80x9d for use in accordance with the invention is the generic name of compounds with 12 members or more, which belong to macrocyclic lactones.
As a particular example of the macrolide compound, the tricyclic compound of the following formula (I) can be exemplified. 
(wherein each of adjacent pairs of R1 and R2, R3 and R4 , and R5 and R6 independently
(a) are two adjacent hydrogen atoms, and R2 may optionally be an alkyl group, or
(b) forms another bond formed between the carbon atoms to which they are attached;
R7 is a hydrogen atom, a hydroxy group, a protected hydroxy group, or an alkoxy group, or an oxo group together with R1;
R8 and R9 are independently a hydrogen atom or a hydroxy group;
R10 is a hydrogen atom, an alkyl group, an alkyl group substituted by one or more hydroxy groups, an alkenyl group, an alkenyl group substituted by one or more hydroxy groups, or an alkyl group substituted by an oxo group;
X is an oxo group, a hydrogen atom and a hydroxy group, (a hydrogen atom and a hydrogen atom), or a group represented by the formula xe2x80x94CH2Oxe2x80x94;
Y is an oxo group, (a hydrogen atom and a hydroxy group), (a hydrogen atom and a hydrogen atom), or a group represented by the formula Nxe2x80x94NR11R12 or Nxe2x80x94OR13;
R11 and R12 are independently a hydrogen atom, an alkyl group, an aryl group or a tosyl group;
R13, R14, R15, R16, R17, R18, R19, R22 and R23 are independently a hydrogen atom or an alkyl group;
R24 is an optionally substituted ring system which may contain one or more heteroatoms;
n is an integer of 1 or 2; and
in addition to the above definitions, Y, R10 and R23, together with the carbon atoms to which they are attached, may represent a saturated or unsaturated 5- or 6-membered nitrogen, sulfur and/or oxygen containing heterocyclic ring optionally substituted by one or more groups selected from the group consisting of an alkyl, a hydroxy, an alkoxy, a benzyl, a group of the formula xe2x80x94CH2Se(C6H5), and an alkyl substituted by one or more hydroxy groups.
The definitions used in the above general formula (I) and the specific and preferred examples thereof are now explained and set forth in detail.
The term xe2x80x9clowerxe2x80x9d means, unless otherwise indicated, a group having 1 to 6 carbon atoms.
Preferable examples of the xe2x80x9calkyl groupsxe2x80x9d and an alkyl moiety of the xe2x80x9calkoxy groupxe2x80x9d include a straight or branched chain aliphatic hydrocarbon residue, for example, a lower alkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, neopentyl and hexyl.
Preferable examples of the xe2x80x9calkenyl groupsxe2x80x9d include a straight or branched chain aliphatic hydrocarbon residue having one double-bond, for example, a lower alkenyl group such as vinyl, propenyl (e.g., allyl group), butenyl, methylpropenyl, pentenyl and hexenyl.
Preferable examples of the xe2x80x9caryl groupsxe2x80x9d include phenyl, tolyl, xylyl, cumenyl, mesityl and naphthyl.
Preferable protective groups in the xe2x80x9cprotected hydroxy groupsxe2x80x9d and the xe2x80x9cprotected aminoxe2x80x9d are 1-(lower alkylthio)-(lower) alkyl group such as a lower alkylthiomethyl group (e.g., methylthiomethyl, ethylthiomethyl, propylthiomethyl, isopropylthiomethyl, butylthiomethyl, isobutylthiomethyl, hexylthiomethyl, etc.), more preferably C1-C4 alkylthiomethyl group, most preferably methylthiomethyl group;
trisubstituted silyl group such as a tri(lower)alkylsilyl (e.g., trimethylsilyl, triethylsilyl, tributylsilyl, tert-butyldimethylsilyl, tri-tert-butylsilyl, etc.) or lower alkyl-diarylsilyl (e.g., methyldiphenylsilyl, ethyldiphenylsilyl, propyldiphenylsilyl, tert-butyldiphenyl-silyl, etc.), more preferably tri(C1-C4)alkylsilyl group and C1-C4 alkyldiphenylsilyl group, most preferably tert-butyldimethylsilyl group and tert-butyldiphenylsilyl group; and an acyl group such as an aliphatic, aromatic acyl group or an aliphatic acyl group substituted by an aromatic group, which are derived from a carboxylic acid, sulfonic acid or carbamic acid.
Examples of the aliphatic acyl groups include a lower alkanoyl group optionally having one or more suitable substituents such as carboxy, e.g., formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, carboxyacetyl, carboxypropionyl, carboxybutyryl, carboxyhexanoyl, etc.; a cyclo(lower)alkoxy(lower)alkanoyl group optionally having one or more suitable substituents such as lower alkyl, e.g., cyclopropyloxyacetyl, cyclobutyloxypropionyl, cycloheptyloxybutyryl, menthyloxyacetyl, menthyloxypropionyl, menthyloxybutyryl, menthyloxypentanoyl, menthyloxyhexanoyl, etc.; a camphorsulfonyl group; or a lower alkylcarbamoyl group having one or more suitable substituents such as carboxy or protected carboxy, for example, carboxy(lower)alkylcarbamoyl group (e.g., carboxymethylcarbamoyl, carboxyethylcarbamoyl, carboxypropylcarbamoyl, carboxybutylcarbamoyl, carboxypentylcarbamoyl, carboxyhexylcarbamoyl, etc.), tri(lower)alkylsilyl(lower)alkoxycarbonyl(lower)alkylcarbamoyl group (e.g., trimethylsilylmethoxycarbonylethylcarbamoyl, trimethylsilylethoxycarbonylpropylcarbamoyl, triethylsilylethoxycarbonylpropylcarbamoyl, tert-butyldimethylsilylethoxycarbonylpropylcarbamoyl, trimethylsilylpropoxycarbonylbutylcarbamoyl, etc.) and so on.
Examples of the aromatic acyl groups include an aroyl group optionally having one or more suitable substituents such as nitro, e.g., benzoyl, toluoyl, xyloyl, naphthoyl, nitrobenzoyl, dinitrobenzoyl, nitronaphthoyl, etc.; and an arenesulfonyl group optionally having one or more suitable substituents such as halogen, e.g., benzenesulfonyl, toluenesulfonyl, xylenesulfonyl, naphthalenesulfonyl, fluorobenzenesulfonyl, chlorobenzenesulfonyl, bromobenzenesulfonyl, iodobenzenesulfonyl, etc.
Examples of the aliphatic acyl groups substituted by an aromatic group include or(lower)alkanoyl group optionally having one or more suitable substituents such as lower alkoxy or trihalo(lower)alkyl, e.g., phenylacetyl, phenylpropionyl, phenylbutyryl, 2-trifluoromethyl-2-methoxy-2-phenylacetyl, 2-ethyl-2-trifluoromethyl-2-phenylacetyl, 2trifluoromethyl-2-propoxy-2-phenylacetyl, etc.
More preferable acyl groups among the aforesaid acyl groups are C1-C4 alkanoyl group optionally having carboxy, cyclo (C5-C6)alkoxy(C1-C4)alkanoyl group having two (C1-C4) alkyls at the cycloalkyl moiety, camphorsulfonyl group, carboxy-(C1-C4)alkylcarbamoyl group, tri(C1-C4)alkylsilyl(C1-C4)alkoxycarbonyl(C1-C4)alkylcarbamoyl group, benzoyl group optionally having one or two nitro groups, benzenesulfonyl group having halogen, or phenyl(C1-C4)alkanoyl group having C1-C4 alkoxy and trihalo(C1-C4)alkyl group. Among these, the most preferable ones are acetyl, carboxypropionyl, menthyloxyacetyl, camphorsulfonyl, benzoyl, nitrobenzoyl, dinitrobenzoyl, iodobenzenesulfonyl and 2-trifluoromethyl-2-methoxy-2-phenylacetyl.
Preferable examples of the xe2x80x9c5- or 6-membered nitrogen, sulfur and/or oxygen containing heterocyclic ringxe2x80x9d include a pyrrolyl group and a tetrahydrofuryl group.
R24 is an optionally substituted ring system which may contain one or more heteroatoms. Preferably R24 may be cyclo(C5-7)alkyl group optionally having suitable substituents, and the following ones can be exemplified.
(a) a 3,4-di-oxo-cyclohexyl group;
(b) a 3-R20-4-R21-cyclohexyl group,
xe2x80x83in which
R20 is hydroxy, an alkoxy group, an oxo group, or
a xe2x80x94OCH2OCH2CH2OCH3 group, and
R21 is hydroxy, xe2x80x94OCN, an alkoxy group, a heteroaryloxy which may be substituted by suitable substituents, 1- or 2-tetrazolyl, a xe2x80x94OCH2OCH2CH2OCH3 group, a protected hydroxy group, chloro, bromo, iodo, aminooxalyloxy, an azido group, p-tolyloxythiocarbonyloxy, or R25R26CHCOOxe2x80x94,
xe2x80x83in which
R25 is optionally protected hydroxy or protected amino, and
R26 R is hydrogen or methyl, or
R20 and R21 together form an oxygen atom in an epoxide ring; or
(c) cyclopentyl group substituted by methoxymethyl, optionally protected hydroxymethyl, acyloxymethyl
(in which the acyl moiety optionally contains either a dimethylamino group which may be quaternized, or a carboxy group which may be esterified), one or more amino and/or hydroxy groups which may be protected, or aminooxalyloxymethyl. A preferred example is a 2-formyl-cyclopentyl group.
xe2x80x9cA heteroaryl which may be substituted by suitable substituentsxe2x80x9d moiety of the xe2x80x9cheteroaryloxy which may be substituted by suitable substituentsxe2x80x9d may be the ones exemplified for R1 of the compound of the formula of EP-A-532,088, with preference given to 1-hydroxyethylindol -5-yl, the disclosure of which is incorporated herein by reference.
The tricyclic compounds (I) and its pharmaceutically acceptable salt for use in accordance with this invention are well known to have excellent immunosuppressive activity, antimicrobial activity and other pharmacological activities and, as such, be of value for the treatment or prevention of rejection reactions by transplantation of organs or tissues, graft-vs-host diseases, autoimmune diseases, and infectious diseases [EP-A-0184162, EP-A-0323042, EP-A-423714, EP-A-427680, EP-A-465426, EP-A-480623, EP-A-532088, EP-A-532089, EP-A-569337, EP-A-626385, WO89/05303, WO93/05058, WO96/31514, WO91/13889, WO91/19495, WO93/04680, WO93/5059, etc.], the disclosures of which are incorporated herein by reference.
Particularly, the compounds which are designated as FR900506 (=FK506), FR900520 (ascomycin), FR900523, and FR900525 are products produced by microorganisms of the genus Streptomyces, such as Streptomyces tsukubaensis No. 9993 [deposited with National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology (formerly Fermentation Research Institute Agency of Industrial Science and Technology ), at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan, date of deposit Oct. 5, 1984, accession number FERM BP-927] or Streptomyces hygroscopius subsp. yakushimaensis No. 7238 [deposited with National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology (formerly Fermentation Research Institute Agency of Industrial Science and Technology ), at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan, date of deposit Jan. 12, 1985, accession number FERM BP-928] [EP-A-0184162]. The FK506 (general name: tacrolimus) of the following chemical formula, in particular, is a representative compound. 
The preferred examples of the tricyclic compounds (I) are the ones, wherein each of adjacent pairs of R3 and R4 or R5 and R6 independently form another bond formed between the carbon atoms to which they are attached;
each of R8 and R23 is independently a hydrogen atom;
R9 is a hydroxy group;
R10 is a methyl group, an ethyl group, a propyl group or an allyl group;
X is (a hydrogen atom and a hydrogen atom) or an oxo group;
Y is an oxo group;
each of R14, R15, R16, R17, R18, R19, and R22 is a methyl group;
R24 is a 3-R20-4-R21-cyclohexyl group,
xe2x80x83in which
R20 is hydroxy, an alkoxy group, an oxo group, or a xe2x80x94OCH2OCH2CH2OCH3 group, and
R21 is hydroxy, xe2x80x94OCN, an alkoxy group, a heteroaryloxy which may be substituted by suitable substituents, 1- or 2-tetrazolyl, a xe2x80x94OCH2OCH2CH2OCH3 group, a protected hydroxy group, chloro, bromo, iodo, aminooxalyloxy, an azido group, p-tolyloxythiocarbonyloxy, or R25R26CHCOOxe2x80x94,
xe2x80x83in which
R25 is optionally protected hydroxy or protected amino, and
R26 is hydrogen or methyl, or
R20 and R21 together form an oxygen atom in an epoxide ring; and
n is an integer of 1 or 2.
The most preferable tricyclic compounds(I) is, in addition to FK506, ascomycin derivatives such as halogenated-ascomycin (e.g., 33-epi-chloro-33-desoxyascomycin), which is disclosed in EP 427,680, example 66a.
The tricyclic compounds(I) has a similar basic structure, i.e., tricyclic macrolide structure, and at least one of the similar biological properties (for example, immunosupressive activity).
The tricyclic compounds(I) may be in a form of its salt, which includes conventional non-toxic and pharmaceutically acceptable salt such as the salt with inorganic or organic bases, specifically, an alkali metal salt such as sodium salt and potassium salt, an alkali earth metal salt such as calcium salt and magnesium salt, an ammonium salt and an amine salt such as triethylamine salt and N-benzyl-N-methylamine salt.
With respect to the macrolide compound used in the present invention, it is to be understood that there may be conformers and one or more stereoisomers such as optical and geometrical isomers due to asymmetric carbon atom(s) or double bond(s), and such conformers and isomers are also included within the scope of macrolide compound in the present invention. And further, the macrolide compounds can be in the form of a solvate, which is included within the scope of the present invention. The solvate preferably include a hydrate and an ethanolate.
The macrolide compounds usable in the present invention may be administered as pure compounds or mixtures of compounds or preferably, in a pharmaceutical vehicle or carrier.
The pharmaceutical compositions of this invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the macrolide compounds of the present invention, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external(topical), enteral, intravenous, intramuscular, or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable, carriers for tablets, pellets, capsules, eye drops, suppositories, solutions (saline, for example), emulsion, suspensions (olive oil, for example), ointment and any other form suitable for use. The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesiumtrisilicate, talc, cornstarch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. The active object compound is included in the pharmaceutical composition in an effective amount sufficient to produce the desired effect upon the process or condition of the disease.
Mammals which may be treated using the method of the present invention include livestock mammals such as cows, horses, etc., domestic animals such as dogs, cats, rats, etc. and humans.
While the dosage of therapeutically effective amount of the macrolide compounds varies from and also depends upon the age and condition of each individual patient to be treated, a daily dose of about 0.0001-1000 mg, preferably, 0.001-500 mg and more preferably, 0.01-100 mg of the active ingredient is generally given for treating diseases, and an average single dose of about 0.001-0.01 mg, 0.2-0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg is generally administered. Daily doses for chronic administration in humans will be in the range of about 0.1-0.3 mg/kg/day.